Conditional superagonist ctl ligands for the promotion of tumor-specific ctl responses

ABSTRACT

What is described is a novel genetic screen, involving recombinant technology and class I antigen cross-presentation, to search for supraoptimal superagonists of the 27L MART-1 mutant selecting for single amino acid substitution mutants of 27L that activate human antigen-specific CTL clones recognizing the wild-type MART-1 26-35  epitope. Three novel mutant epitopes are identified with superagonist properties that are functionally superior to 27L. The ability of a given analog to act as superagonist varies among patients. Also described is the use of methods to establish panels of potential superagonist APLs to individualize tumor peptide vaccines among patients. The methodology is replicated to identify APL to NY-ESO-1 157-165  and NY-ESO-1 157-170  tumor epitopes. A general method is described that is useful to produce a tumor vaccine to any tumor epitope.

TECHNICAL FIELD

What is described is a method of identifying antigens for a cancer vaccine, and specific polypeptides and polynucleotides useful in producing vaccines against cells having NY-ESO-1₁₅₇₋₁₆₅, NYE-ESO-1₁₅₇₋₁₇₀, or MART-1₂₆₋₃₅ tumor epitopes.

BACKGROUND

Cytotoxic T lymphocytes can directly kill malignant cells, which express and display specific antigenic peptides in the context of specific class I MHC molecules. These antigenic peptides, often referred to as CTL epitopes, are peptides of unique amino acid sequence, usually 9-11 amino acids in length. The tumor-associated antigenic peptide that is being targeted can be used as a peptide-based vaccine to promote the anti-tumor CTL response. However, when the target peptide is derived from non-mutated differentiation antigens as is often the case (e.g. melanosomal proteins), it can be insufficient to engender robust and sustained anti-tumor CTL responses. This is a result of immune tolerance mechanisms that generally suppress or eliminate high avidity auto-reactive T cells. As a result of these mechanisms, the vast majority of tumor-specific CTL, specifically those that recognize non-mutated tumor-associated antigens, are eliminated in the thymus and in the periphery. What remains is a low frequency of tumor-specific CTL, and/or CTL that bear low avidity T cell receptors for the cognate tumor antigen.

One way to activate and mobilize these rare and low avidity tumor-specific CTL is with the use of superagonist altered peptide ligands (APLs). These are mutant peptide ligands that deviate from the native peptide sequence by one or more amino acids, and which activate specific CTL clones more effectively than the native epitope. These alterations either allow the peptide to bind better to the restricting class I MHC molecule or interact more favorably with the TCR of a given tumor-specific CTL subset. Superagonist APLs demonstrate favorable responses in clinical studies.

One method to identify superagonist APLs involves comparing the amino acid sequence of the tumor-associated CTL epitope to the so-called consensus binding motif for the restricting class I MHC allotype. Where the tumor-associated epitope deviates from the consensus sequence, the appropriate amino acids can be substituted, allowing the peptide to bind better to the class I MHC molecule. This approach is limited because not all poorly stimulatory CTL epitopes deviate from the consensus motif. Another approach involves substituting one or more specific amino acids into every position of the epitope; e.g., alanine scanning. Another approach includes making every single amino acid substitution at one or two positions—positions either predicted to play a role in class I MHC secondary binding or to be directly involved in engaging the TCR. All of these approaches are severely limited in scope, and potentially overlook a large number of superagonist APLs. Utilization of APLs remains limited due to a lack of comprehensive methods for which to identify them.

SUMMARY

One aspect of the invention is a method of identifying an altered peptide ligand (APL) for eliciting response of regulator or effector CD4 or CD8 T cells against a tumor epitope, consisting of the steps of:

-   -   i. preparing saturation mutagenesis oligonucleotides encoding         APLs;     -   ii. cloning said oligonucleotides in an expression vector;     -   iii. separating APL expressed by a clone from other cellular         proteins;     -   iv. treat the T cells with the APL; and     -   v. identifying a superagonist APL that maximally stimulates the         T cells.

An embodiment of the method, is a further step of screening the APLs for the ability to activate epitope-specific CTL clones. Another embodiment is the method in which the APLs are cross-presented to CTL clones on class I MHC molecules by immature dendritic cells. Another embodiment is the method wherein the oligonucleotides are cloned into bacteria. Another embodiment is wherein the bacteria are grown in 5 ml cultures, preferably less than 1 ml and preferably more than 0.2 ml. Another is the method, wherein the APL stimulates T cells to produce interferon-γ.

Another aspect of the method is wherein the tumor epitope is selected from the group consisting of SEQ ID NOS:1-351. An embodiment of the method is wherein the tumor epitope is NY-ESO-1₁₅₇₋₁₆₅ (SEQ ID NO:366), NY-ESO-1₁₅₇₋₁₇₀ (SEQ ID NO:367) or MART-1₂₆₋₃₅ (SEQ ID NO:361). Another embodiment is the superagonist APL identified by use of these tumor epitopes. Another embodiment is the method, wherein a panel of superagonist APLs are identified.

Another aspect of the invention is the superagonist APL identified according to the method, including SEQ ID NOS:362-365 and 368-375. One or more APL is useful as a component in an antitumor vaccine. An embodiment is a panel of superagonist APLs, consisting of two or more APLs.

Another aspect of the invention is a method of using the superagonist APL by combining it with cells of a patient. An embodiment is a method by which the superagonist APL is administered to the patient. In this context, the superagonist APL may be used in an anti-tumor vaccine, in adoptive immunotherapy to generate T cell clonotypes, and/or to alter the phenotype of regulatory T cells to more effectively activate anti-tumor T cells. This embodiment of the invention may involve cells of the patient being treated ex vivo.

Another aspect of the invention is the method of stimulating T cells with an APL or APL panel, wherein the CD4 T cells are one or more T cells selected from the group consisting of Th1, Th2, Th9, and Th17 cells.

Another aspect of the invention is a superagonist APL selected from the group consisting of SEQ:362-365 and 368-376. An embodiment of the invention is a panel of superagonist APLs selected from the group consisting of SEQ:362-365 and 368-376.

Another aspect of the invention is an APL minigene, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:382-391 and 377-381. An embodiment of the invention is a use of the APL minigene to express the minigene in a human cell, preferably a cell of a patient, most preferably, a cell from a patient with a cancer or cancer precursor, a graft versus host disease, or an autoimmune condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Native and superagonist CTL determinants can be distinguished in bead-based cross-presentation assay. Oligonucleotides encoding MART-1₂₆₋₃₅ (SEQ ID NO:361), NY-ESO-1₁₅₇₋₁₆₅ (SEQ ID NO:366), or MART-1₂₆₋₃₅A27L (“A27L,” SEQ ID NO:362) were cloned into and expressed by pQE40 expression vectors in 5 ml bacterial cultures. The mini-gene products were isolated and “fed” to immature dendritic cells as described in the Example 8. MART-1₂₆₋₃₅-specific CTL clones were used to detect the presence of the cross-presented mini-gene products. Induced IFN-γ expression was determined by standard sandwich ELISA. A27L synthetic peptide at 1 μM was used a positive control.

FIG. 2. Previously described superagonists identified in MART-1₂₆₋₃₅ Position 2 saturation mutagenesis APL screen. 88 P2 saturation mutagenesis clones were screened. MART-1₂₆₋₃₅ control construct is the first bar on left, and the NY-ESO-1₁₅₇₋₁₆₅ negative control construct is the second bar from left. APL clones eliciting comparable IFN-γ expression as the native construct were sequenced. The amino acid at position 27 is shown above for the most active polypeptide sequences.

FIG. 3. Eight positional libraries of A27L were screened using the saturation mutagenesis technique. 88 mutant clones were screened for each of eight positional libraries of A27L—P1, P3, P4, P5, P6, P7, P8 and P9. (A) Two clones are screened simultaneously for each library. Activation was assessed by IFN-γ expression. Positive control (A27L) is the far left bar while the negative control (NY-ESO-1₁₅₇₋₁₆₅) is the second from left. APL clonal wells indicated with an arrow were de-convoluted and each mutant APL re-screened separately. The table (B) shows the IFN-γ activity elicited by individual clones, relative to the activity elicited by A27L. The clones that were initially assayed together are indicated by shading. A bold number indicates the APL clone which is most responsible for the activation of the screening CTL clone. DNA sequence analysis was used to determine the amino acid encoded.

FIG. 4. APLs identified in saturation mutagenesis screen activate unique MART-1₂₆₋₃₅-specific CTL clones differently. Two unique high avidity MART-1₂₆₋₃₅-specific CTL clones, M26-H1 (A) and M26-H2 (B), and two unique low avidity MART-1₂₆₋₃₅-specific CTL clones, M26-L1(C) and M26-L2 (D), were assayed against the agonist peptides A27L (square), E26G (SEQ ID NO:363; circle), E26S (SEQ ID NO:364; triangle), L33M (SEQ ID NO:365; diamond) and NY-ESO-1₁₅₇₋₁₆₅ (x's). Peptides were titrated on T2 target cell. IFN-γ expression was measured by standard ELISA.

FIG. 5. APLs generate different CTL responses from the PBMC of different melanoma patients. Identified APLs (Example 12) were used to stimulate the PBMC of different melanoma patients in vitro. Following a one week primary and one week secondary peptide stimulation, cultures were stained with FITC-labeled anti-CD8 antibody and APC-labeled HLA-A2/MART-1₂₆₋₃₅ tetramer and analyzed by flow cytometry. Data is representative of at least three different experiments.

FIG. 6. Native and superagonist CTL determinants can be distinguished in bead-based cross-presentation assay. Oligonucleotides encoding NY-ESO-1₁₅₇₋₁₇₀ were cloned into and expressed by pQE40 expression vectors. The mini-gene products were isolated and “fed” to immature dendritic cells as described in Examples 2-8. NY-ESO-1₁₅₇₋₁₇₀₋specific CTL clones were used to detect the presence of the cross-presented mini-gene products. Induced IFN-γ expression was determined by standard sandwich ELISA. Synthetic wild-type peptide was used a positive control.

FIG. 7. NY-ESO-1157-165 9C->9V generates Specific CTL better than the wild type peptide. Following a 1-week primary and 1-week secondary peptide stimulation (Week 2), and an additional week (Week 3), cultures were stained with FITC-labeled anti-CD8 antibody and APC-labeled HLA-A2/NY-ESO and analyzed by flow cytometry. Two peptides were tested, NY-ESO-1₁₅₇₋₁₆₅ (SEQ ID NO:366) and NY-ESO-1₁₅₇₋₁₆₅ (V) (SEQ ID NO:376).

FIG. 8. Native and superagonist CTL determinants can be distinguished in bead-based cross-presentation assay. Oligonucleotides encoding NY-ESO-1₁₅₇₋₁₇₀ were cloned into and expressed by pQE40 expression vectors. The mini-gene products were isolated and “fed” to immature dendritic cells as described in Examples 2-8. NY-ESO-1₁₅₇₋₁₇₀-specific CTL clones were used to detect the presence of the cross-presented mini-gene products (NY-ESO-1₁₅₇₋₁₇₀). PBMC (FIG. 8B) were compared to CD-4⁺ cells (FIG. 8A). Induced IFN-γ expression was determined by standard sandwich ELISA. Synthetic wild-type peptide was used a positive control.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

What is described herein is a method to screen for potential superagonist APLs of a clinically relevant tumor-associated antigen, including NY-ESO-1 and MART-1. Rather than screening a limited subset of possible agonists, this technique allows screening of every single amino acid mutant of tumor epitope in a rapid and cost-effective manner. This approach to identifying APLs is effective, given the difference even subtle amino acid substitutions have on specific T cell response. Since superagonist APL structure cannot be predicted, the method described generates candidate APLS by a comprehensive screening technique.

Another aspect of unpredictability is that a given agonist APL may be more or less effective for different patients. While a given agonist APL might have a high stimulatory capacity for one patient it could be relatively ineffective for another patient. Apparently, different clones are being mobilized with different agonist peptides. This heightens the need for panels of superagonist APLs for use in a therapeutic setting.

Tumor-Specific Epitopes

Unique antigens result from point mutations in genes that are expressed ubiquitously. The mutation usually affects the coding region of the gene and is unique to the tumor of an individual patient or restricted to very few patients. Antigens that are strictly tumor-specific may play an important role in the natural anti-tumor immune response of individual patients. These are listed in Table 1.

These epitopes are characteristic of lung carcinoma, melanoma, chronic myeloid leukemia, colorectal carcinoma, gastric carcinoma, endometrial carcinoma, head and neck squamous cell carcinoma, lung squamous cell carcinoma, renal cell carcinoma, bladder tumor, non-small cell lung carcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, sarcoma, promyelocytic leukemia, myeloid leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, breast cancer, and prostate carcinoma.

Each epitope is associated with a particular HLA haplotype, either a class I or class II MHC antigen, as shown in Tables 1-4.

TABLE 1 Unique antigens SEQ ID Gene/protein HLA haplotype Peptide^(c) Position NO alpha-actinin-4 A2 FIASNGVKLV  118-127 1 ARTC1 DR1 YSVYFNLPADTIYTN 2 BCR-ABL fusion protein A2 SSKALQRPV  926-934 3 (b3a2) B8 GFKQSSKAL  922-930 392 DR4 ATGFKQSSKALQRPVAS  920-936 393 DR9 ATGFKQSSKALQRPVAS  920-936 394 B-RAF DR4 EDLTVKIGDFGLATEKSRWSGSHQFEQLS  586-614 4 CASP-5 A2 FLIIWQNTM   67-75 5 CASP-8 B35 FPSDSWCYF  476-484 6 beta-catenin A24 SYLDSGIHF   29-37 7 Cdc27 DR4 FSWAMDLDPKGA  760-771 8 CDK4 A2 ACDPHSGHFV   23-32 9 CDKN2A A11 AVCPWTWLR  125-133 10 (p14ARF-ORF3)  111-119 (p16INK4a- ORF3) COA-1 DR4 TLYQDDTLTLQAAG  371-384 11 DR13 TLYQDDTLTLQAAG  371-384 dek-can fusion protein DR53 TMKQICKKEIRRLHQY  342-357 12 EFTUD2 A3 KILDAVVAQK  668-677 13 Elongation factor 2 A68 ETVSEQSNV  581-589 14 ETV6-AML1 A2 RIAECILGM  334-342 15 fusion protein DP5 IGRIAECILGMNPSR  332-346 16 DP17 IGRIAECILGMNPSR  332-346 16 FLT3-ITD A1 YVDFREYEYY  591-600 17 FN1 DR2 MIFEKHGFRRTTPP 2050-2063 18 GPNMB A3 TLDWLLQTPK  179-188 19 LDLR-fucosyltransferase DR1 WRRAPAPGA  315-323 20 AS fusion protein DR1 PVTWRRAPA  312-320 21 hsp70-2 A2 SLFEGIDIYT  286-295 22 KIAAO205 B44 AEPINIQTW  262-270 23 MART2 A1 FLEGNEVGKTY  446-455 24 ME1 A2 FLDEFMEGV  224-232 25 MUM-1^(f) B44 EEKLIVVLF   30-38 26 MUM-2 B44 SELFRSGLDSY  123-133 27 Cw6 FRSGLDSYV  126-134 28 MUM-3 A68 EAFIQPITR  322-330 29 neo-PAP DR7 RVIKNSIRLTL  724-734 30 Myosin class I A3 KINKNPKYK  911-919 31 NFYC B52 QQITKTEV  275-282 32 OGT A2 SLYKFSPFPL   28-37 33 OS-9 B44 KELEGILLL  438-446 34 p53 A2 VVPCEPPEV  217-225 35 pml-RAR alpha fusion DR11 NSNHVASGAGEAAIETQSSSSEEIV 36 protein PRDX5 A2 LLLDDLLVSI  163-172 37 PTPRK DR10 PYYFAAELPPRNLPEP  667-682 38 K-ras B35 VVVGAVGVG    7-15 39 N-ras A1 ILDTAGREEY   55-64 40 RBAF600 B7 RPHVPESAF  329-337 41 SIRT2 A3 KIFSEVTLK  192-200 42 SNRPD1 B38 SHETVIIEL   11-19 43 SYT-SSX1 or -SSX2 B7 QRPYGYDQIM  402-410 (SYT) 44 fusion protein  111-112(SSX2) TGF-betaRII A2 RLSSCVPVA  131-139 45 Triosephosphate DR1 GELIGILNAAKVPAD   23-37 46 isomerase

Shared antigens are present on many independent tumors. One group corresponds to peptides encoded by “cancer-germline” genes that are expressed in many tumors but not in normal tissues. Some are listed in Table 2.

TABLE 2 Shared tumor-specific antigens Gene HLA Peptide Position SEQ ID NO: 4BAGE-1 Cw16 AARAVFLAL   2-10 47 GAGE-1,2,8 Cw6 YRPRPRRY   9-16 48 GAGE-3,4,5,6,7 A29 YYWPRPRRY  10-18 49 GnTV^(f) A2 VLPDVFIRC(V) intron 50 HERV-K-MEL A2 MLAVISCAV   1-9 51 KK-LC-1 B15 RQKRILVNL  76-84 52 KM-HN-1 A24 NYNNFYRFL 196-204 53 A24 EYSKECLKEF 499-508 54 A24 EYLSLSDKI 770-778 55 LAGE-1 A2 MLMAQEALAFL ORF2 56 (1-11) A2 SLLMWITQC 157-165 57 A31 LAAQERRVPR ORF2 58 (18-27) A68 ELVRRILSR 103-111 59 B7 APRGVRMAV ORF2 60 (46-54) DP4 SLLMWITQCFLPVF 157-170 61 DR3 QGAMLAAQERRVPRAAEVPR ORF2 62 (14-33) DR4 AADHRQLQLSISSCLQQL 139-156 63 DR11 CLSRRPWKRSWSAGSCPGMPHL ORF2 64 (81-102) DR12 CLSRRPWKRSWSAGSCPGMPHL ORF2 65 (81-102) DR13 ILSRDAAPLPRPG 108-120 66 DR15 AGATGGRGPRGAGA  37-50 67 MAGE-A1 A1 EADPTGHSY 161-169 68 A2 KVLEYVIKV 278-286 69 A3 SLFRAVITK  96-104 70 A68 EVYDGREHSA 222-231 71 B7 RVRFFFPSL 289-298 72 B35 EADPTGHSY 161-169 73 B37 REPVTKAEML 120-129 74 B53 DPARYEFLW 258-266 75 B57 ITKKVADLVGF 102-112 76 Cw2 SAFPTTINF  62-70 77 Cw3 SAYGEPRKL 230-238 78 Cw16 SAYGEPRKL 230-238 79 DP4 TSCILESLFRAVITK  90-104 80 DP4 PRALAETSYVKVLEY 268-282 81 DR13 FLLLKYRAREPVTKAE 112-127 82 DR15 EYVIKVSARVRF 281-292 83 MAGE-A2 A2 YLQLVFGIEV 157-166 84 A24 EYLQLVFGI 156-164 85 B37 REPVTKAEML 127-136 86 Cw7 EGDCAPEEK 212-220 87 DR13 LLKYRAREPVTKAE 121-134 88 MAGE-A3 A1 EVDPIGHLY 168-176 89 A2 FLWGPRALV 271-279 90 A2 KVAELVHFL 112-120 91 A24 TFPDLESEF  97-105 92 A24 VAELVHFLL 113-121 93 B18 MEVDPIGHLY 167-176 94 B35 EVDPIGHLY 168-176 95 B37 REPVTKAEML 127-136 96 B40 AELVHFLLL 114-122 97 B44 MEVDPIGHLY 167-176 98 B52 WQYFFPVIF 143-151 99 Cw7 EGDCAPEEK 212-220 100 DP4 KKLLTQHFVQENYLEY 243-258 101 DQ6 KKLLTQHFVQENYLEY 243-258 102 DR1 ACYEFLWGPRALVETS 267-282 103 DR4 VIFSKASSSLQL 149-160 104 DR7 VIFSKASSSLQL 149-160 105 DR11 GDNQIMPKAGLLIIV 191-205 106 DR11 TSYVKVLHHMVKISG 281-295 107 DR13 RKVAELVHFLLLKYRA 111-126 108 DR13 FLLLKYRAREPVTKAE 119-134 109 MAGE-A4 A1 EVDPASNTY 169-177 110 A2 GVYDGREHTV 230-239 111 A24 NYKRCFPVI 143-151 112 B37 SESLKMIF 156-163 113 MAGE-A6 A34 MVKISGGPR 290-298 114 B35 EVDPIGHVY 168-176 115 B37 REPVTKAEML 127-136 116 Cw7 EGDCAPEEK 212-220 117 Cw16 ISGGPRISY 293-301 118 DR13 LLKYRAREPVTKAE 121-134 119 MAGE-A9 A2 ALSVMGVYV 223-231 120 MAGE-A10 A2 GLYDGMEHL 254-262 121 B53 DPARYEFLW 290-298 122 MAGE-A12 A2^(g) FLWGPRALV 271-279 123 Cw7 VRIGHLYIL 170-178 124 Cw7 EGDCAPEEK 212-220 125 DP4 REPFTKAEMLGSVIR 127-141 126 DR13 AELVHFLLLKYRAR 114-127 127 MAGE-C2 A2 LLFGLALIEV 191-200 128 A2 ALKDVEERV 336-344 129 B44 SESIKKKVL 307-315 130 mucin ^(k) PDTRPAPGSTAPPAHGVTSA 131 NA88-A B13 QGQHFLQKV 132 NY-ESO-1/LAGE-2 A2 SLLMWITQC 157-165 133 A2 MLMAQEALAFL ORF2 134 (1-11) A31 ASGPGGGAPR  53-62 135 A31 LAAQERRVPR ORF2 136 (18-27) A68 TVSGNILTIR 127-136 137 B7 APRGPHGGAASGL  60-72 138 B35 MPFATPMEA  94-102 139 B49 KEFTVSGNILTI 124-135 140 B51 MPFATPMEA  94-102 141 Cw3 LAMPFATPM  92-100 142 Cw6 ARGPESRLL  80-88 143 DP4 SLLMWITQCFLPVF 157-170 144 DP4 LLEFYLAMPFATPMEAELARRSLAQ  87-111 145 DR1 LLEFYLAMPFATPMEAELARRSLAQ  87-111 146 DR1 EFYLAMPFATPM  89-100 147 DR2 RLLEFYLAMPFA  86-97 148 DR3 QGAMLAAQERRVPRAAEVPR ORF2 149 (14-33) DR4 PGVLLKEFTVSGNILTIRLT 119-138 150 DR4 VLLKEFTVSG 121-130 151 DR4 AADHRQLQLSISSCLQQL 139-156 152 DR4 LLEFYLAMPFATPMEAELARRSLAQ  87-111 153 DR7 PGVLLKEFTVSGNILTIRLTAADHR 119-143 154 DR7 LLEFYLAMPFATPMEAELARRSLAQ  87-111 155 DR15 AGATGGRGPRGAGA  37-50 156 SAGE A24 LYATVIHDI 715-723 157 Sp17 A1 ILDSSEEDK 103-111 158 SSX-2 A2 KASEKIFYV  41-49 159 DP1 EKIQKAFDDIAKYFSK  19-34 160 DR3 WEKMKASEKIFYVYMKRK  37-54 161 DR4 KIFYVYMKRKYEAMT  45-59 162 DR11 KIFYVYMKRKYEAM  45-58 163 SSX-4 DP10 INKTSGPKRGKHAWTHRLRE 151-170 164 DR3 YFSKKEWEKMKSSEKIVYVY  31-50 165 DR8 MKLNYEVMTKLGFKVTLPPF  51-70 166 DR8 KHAWTHRLRERKQLVVYEEI 161-180 167 DR11 LGFKVTLPPFMRSKRAADFH  61-80 168 DR15 KSSEKIVYVYMKLNYEVMTK  41-60 169 DR52 KHAWTHRLRERKQLVVYEEI 161-180 170 TAG-1 A2 SLGWLFLLL  78-86 171 B8 LSRLSNRLL  42-50 172 TAG-2 B8 LSRLSNRLL  42-50 173 TRAG-3 DR1 CEFHACWPAFTVLGE  34-48 174 DR4 CEFHACWPAFTVLGE  34-48 175 DR7 CEFHACWPAFTVLGE  34-48 176 TRP2-INT2^(g) A68 EVISCKLIKR intron 2 177 XAGE-1b DR9 CATWKVICKSCISQTPG  33-49 178

A second group of shared tumor antigens, named differentiation antigens, are also expressed in the normal tissue of origin of the malignancy. Antigens of this group are not tumor-specific, and their use as targets for cancer immunotherapy may result in autoimmunity towards the corresponding normal tissue. Autoimmune toxicity should not be an issue, however, in situations where the tissue expressing the antigen is dispensable or even resected by the surgeon in the course of cancer therapy, as would be the case for prostate specific antigen (PSA). These antigens are listed in Table 3.

TABLE 3 Differentiation antigens Gene/protein HLA^(a) Peptide Position SEQ ID NO: CEA A2 YLSGANLNL 605-613 179 A2 IMIGVLVGV 691-699 180 A2 GVLVGVALI 694-702 181 A3 HLFGYSWYK  61-69 182 A24 QYSWFVNGTF 268-277 183 A24 TYACFVSNL 652-660 184 DR3 AYVCGIQNSVSANRS 568-582 185 DR4 DTGFYTLHVIKSDLVNEEATGQFRV 116-140 186 DR4 YSWRINGIPQQHTQV 625-639 187 DR7 TYYRPGVNLSLSC 425-437 188 DR7 EIIYPNASLLIQN  99-111 189 DR9 YACFVSNLATGRNNS 653-667 190 DR11 LWWVNNQSLPVSP 177-189 191 and 355-367 DR13 LWWVNNQSLPVSP 177-189 192 and 355-367 DR14 LWWVNNQSLPVSP 177-189 193 and 355-367 DR14 EIIYPNASLLIQN  99-111 194 DR14 NSIVKSITVSASG 666-678 195 gp100/Pmel17 A2 KTWGQYWQV 154-162 196 A2 (A)MLGTHTMEV 177(8)-186 197 A2 ITDQVPFSV 209-217 198 A2 YLEPGPVTA 280-288 199 A2 LLDGTATLRL 457-466 200 A2 VLYRYGSFSV 476-485 201 A2 SLADTNSLAV 570-579 202 A2 RLMKQDFSV 619-627 203 A2 RLPRIFCSC 639-647 204 A3 LIYRRRLMK 614-622 205 A3 ALLAVGATK  17-25 206 A3 IALNFPGSQK  86-95 207 A3 ALNFPGSQK  87-95 208 A11 ALNFPGSQK  87-95 209 A24 VYFFLPDHL intron 4 210 A32 RTKQLYPEW  40-42 211 and  47-52 A68 HTMEVTVYHR 182-191 212 B7 SSPGCQPPA 529-537 213 B35 VPLDCVLYRY 471-480 214 B35 LPHSSSHWL 630-638 215 Cw8 SNDGPTLI  71-78 216 DQ6 GRAMLGTHTMEVTVY 175-189 217 DR4 WNRQLYPEWTEAQRLD  44-59 218 DR7 TTEWVETTARELPIPEPE 420-437 219 DR7 TGRAMLGTHTMEVTVYH 174-190 220 DR53 GRAMLGTHTMEVTVY 175-189 221 Kallikrein 4 DP4 SVSESDTIRSISIAS 125-139 222 DR4 LLANGRMPTVLQCVN 155-169 223 DR7 RMPTVLQCVNVSVVS 160-174 224 mammaglobin-A A3 PLLENVISK  23-31 225 Melan-A/MART-1 A2 (E)AAGIGILTV 26(27)-35 226 A2 ILTVILGVL  32-40 227 B35 EAAGIGILTV  26-35 228 B45 AEEAAGIGIL(T) 24-33(34) 229 Cw7 RNGYRALMDKS  51-61 230 DQ6 EEAAGIGILTVI  25-36 231 DR1 AAGIGILTVILGVL  27-40 232 DR3 EEAAGIGILTVI  25-36 233 DR4 RNGYRALMDKSLHVGTQCALTRR  51-73 234 DR11 MPREDAHFIYGYPKKGHGHS   1-20 235 DR52 KNCEPVVPNAPPAYEKLSAE  91-110 236 NY-BR-1 A2 SLSKILDTV 904-912 237 OA1 A24 LYSACFWWL 126-134 238 PSA A2 FLTPKKLQCV 165-174 239 A2 VISNDVCAQV 178-187 240 RAB38/NY-MEL-1 A2 VLHWDPETV  50-58 241 TRP-1/gp75 A31 MSLQRQFLR alt. ORF 242 DR4 ISPNSVFSQWRVVCDSLEDYD 277-297 243 DR15 SLPYWNFATG 245-254 244 TRP-2 A2 SVYDFFVWL 180-188 245 A2 TLDSQVMSL 360-368 246 A31 LLGPGRPYR 197-205 247 A33 LLGPGRPYR 197-205 248 Cw8 ANDPIFVVL 387-395 249 DR3 QCTEVRADTRPWSGP  60-74 250 DR15 ALPYWNFATG 241-250 251 tyrosinase A1 KCDICTDEY 243-251 252 A1 SSDYVIPIGTY 146-156 253 A2 MLLAVLYCL   1-9 254 A2 CLLWSFQTSA   8-17 255 A2 YMDGTMSQV 369-377 256 A24 AFLPWHRLF 206-214 257 A26 QCSGNFMGF  90-98 258 B35 TPRLPSSADVEF 309-320 259 B35 LPSSADVEF 312-320 260 B38 LHHAFVDSIF 388-397 261 B44 SEIWRDIDF 192-200 262 DR4 QNILLSNAPLGPQFP  56-70 263 DR4 SYLQDSDPDSFQD 450-462 264 DR15 FLLHHAFVDSIFEQWLQRHRP 386-406 265

Shared antigens of the third group are expressed in a wide variety of normal tissues and overexpressed in tumors. Because a minimal amount of peptide is required for CTL recognition, a low level of expression in normal tissues may mean that autoimmune damage is not incurred. However, this threshold is difficult to define, as is the normal level of expression of those genes for each cell type. A list of these is in Table 4.

TABLE 4 Overexpressed antigens Gene HLA^(a) Peptide Position SEQ ID NO: adipophilin A2 SVASTITGV  129-137 266 AIM-2 A1 RSDSGQQARY intron 267 ALDH1A1 A2 LLYKLADLI   88-96 268 BCLX (L) A2 YLNDHLEPWI  173-182 269 BING-4 A2 CQWGRLWQL ORF2 270 CALCA A2 VLLQAGSLHA   16-25 271 CPSF A2 KVHPVIWSL  250-258 272 A2 LMLQNALTTM 1360-1369 273 cyclin D1 A2 LLGATCMFV  101-109 274 DR4 NPPSMVAAGSVVAAV  198-212 275 DKK1 A2 ALGGHPLLGV   20-29 276 ENAH (hMena) A2 TMNGSKSPV  502-510 277 Ep-CAM A24 RYQLDPKFI  173-181 278 EphA3 DR11 DVTFNIICKKCG  356-367 279 EZH2 A2 FMVEDETVL  120-128 280 A2 FINDEIFVEL  165-174 281 A24 KYDCFLHPF  291-299 282 A24 KYVGIEREM  735-743 283 FGF5 A3 NTYASPRFK  172-176 284 and  204-207 G250/MN/CAIX A2 HLSTAFARV  254-262 285 HER-2/neu A2 KIFGSLAFL  369-377 286 A2 IISAVVGIL  654-662 287 A2 ALCRWGLLL    5-13 288 A2 ILHNGAYSL  435-443 289 A2 RLLQETELV  689-697 290 A2 VVLGVVFGI  665-673 291 A2 YMIMVKCWMI  952-961 292 A2 HLYQGCQVV   48-56 293 A2 YLVPQQGFFC 1023-1032 294 A2 PLQPEQLQV  391-399 295 A2 TLEEITGYL  402-410 296 A2 ALIHHNTHL  466-474 297 A2 PLTSIISAV  650-658 298 A3 VLRENTSPK  754-762 299 A24 TYLPTNASL   63-71 300 IL13Ralpha2 A2 WLPFGFILI  345-353 301 Intestinal carboxyl esterase B7 SPRWWPTCL alt. ORF 302 alpha-fetoprotein A2 GVALQTMKQ  542-550 303 A2 FMNKFIYEI  158-166 304 DR13 QLAVSVILRV  364-373 305 M-CSF B35 LPAVVGLSPGEQEY alt. ORF 306 MCSP DR11 VGQDVSVLFRVTGALQ  693-708 307 mdm-2 A2 VLFYLGQY   53-60 308 Meloe A2 TLNDECWPA   36-44 309 MMP-2 A2 GLPPDVQRV  560-568 310 MMP-7 A3 SLFPNSPKWTSK   96-107 311 MUC1 A2 STAPPVHNV  950-958 312 A2 LLLLTVLTV   12-20 313 DR3 PGSTAPPAHGVT repeated region 314 p53 A2 LLGRNSFEV  264-272 315 A2 RMPEAAPPV   65-73 316 B46 SQKTYQGSY   99-107 317 DP5 PGTRVRAMAIYKQ  153-165 318 DR14 HLIRVEGNLRVE  193-204 319 PAX5 A2 TLPGYPPHV  311-319 320 PBF B55 CTACRWKKACQR  499-510 321 PRAME A2 VLDGLDVLL  100-108 322 A2 SLYSFPEPEA  142-151 323 A2 ALYVDSLFFL  300-309 324 A2 SLLQHLIGL  425-433 325 A24 LYVDSLFFL  301-309 326 PSMA A24 NYARTEDFF  178-186 327 RAGE-1 A2 LKLSGVVRL  352-360 328 A2 PLPPARNGGL   32-40 329 B7 SPSSNRIRNT   11-20 330 RGS5 A2 LAALPHSCL    5-13 331 A3 GLASFKSFLK   74-83 332 RhoC A3 RAGLQVRKNK  176-185 333 RNF43 A2 ALWPWLLMA(T) 11-19(20) 334 A24 NSQPVWLCL  721-729 335 RU2AS B7 LPRWPPPQL antisense 336 secernin 1 A2 KMDAEHPEL  196-204 337 SOX10 A2 AWISKPPGV  332-340 338 A2 SAWISKPPGV  331-340 339 STEAP1 A2 MIAVFLPIV  292-300 340 A2 HQQYFYKIPILVINK  102-116 341 survivin A2 ELTLGEFLKL   95-104 342 Telomerase A2 ILAKFLHWL  540-548 343 A2 RLVDDFLLV  865-873 344 DR7 RPGLLGASVLGLDDI  672-686 345 DR11 LTDLQPYMRQFVAHL  766-780 346 VEGF B27 SRFGGAVVR —^(i) 347 WT1 A1 TSEKRPFMCAY  317-327 348 A24 CMTWNQMNL  235-243 349 DP5 LSHLQMHSRKH  337-347 350 DR4 KRYFKLSHLQMHSRKH  332-347 351

Mutagenesis

After selecting the particular tumor specific epitope, random amino acid substitutions are introduced. Oligonucleotide sequences encoding the peptide epitope are designed and cloned in an appropriate vector. Mutagenesis can be done according to the skill of the ordinary worker at each amino acid position of the peptide. The mutant may have substitutions at 1, 2, 3, 4, 5, 6 or more positions, depending on the particular epitope.

The positional libraries are designed such that the codon of interest is totally randomized (NNN), resulting in a pool of oligonucleotides which contains every given codon sequence. This mutagenesis approach might be likened to a slot machine which contains three positions (a codon) and each position has the same 4 possibilities (A, C, G, or T). When pulled, there is a 1 in 64 chance of getting any combination of 3. If pulled 100 times there is a high probability that every sequence will be represented (80% certainty, according to a Poisson distribution). Here, the 100 pulls represent 100 bacterial colonies, each containing a different mutant agonist peptide-encoding oligonucleotide. When cloned and expressed, each amino acid should be represented in a library of 100, with 80% certainty, according to a Poisson distribution. A positional library can be generated for each position (amino acid) of the target peptide. The APL minigene constructs are fused to a 6×-histidine tag, and can easily be separated from bacterial proteins on Co²⁺-coated paramagnetic beads.

The mutagenized epitopes are preferentially expressed in cells as part of an expression vector, more preferentially as a fusion protein. The preferred host for the expression vector is bacterial, e.g., a strain of E. coli. Most preferred is an inducible expression system.

A mutant library is generated using the expression vector in the host cell. Preferentially, the library is distributed in liquid culture, most preferentially in 96 well plates. The cells accumulate a recombinant protein comprising the sequence of the mutagenized epitopes.

The recombinant protein is released and separated from the host cells. This can be done by lysing the cells to release the recombinant protein. Preferentially, the mutagenized epitope is separated from other cellular proteins by adding protein binding magnetic beads (e.g. 6×-histidine specific magnetic beads) to cell lysates.

Screening

Initial screens can be done by combining beads containing recombinant mutagenized epitopes with dendritic cells and epitope-specific T cells and assaying for the production appropriate cytokines, including, but not limited to, interferon γ, interleukin-4, interleukin-10, and granulocyte macrophage colony-stimulating factor. That is, APLs are screened for the ability to activate epitope-specific T cell clones following cross-presentation of the bead-bound ligand on class I or class II MHC molecules by dendritic cells (DC).

Attempts by others to measure the functional avidity of tumor epitope-specific CTL generated via unmodified peptide with CTL generated via the analogs, have been hampered by the inability to generate CD8⁺/MART-1₂₆₋₃₅-tetramer positive T cell populations using a peptide having the natural amino acid sequence of the epitope. Using the methods described herein, the superagonist APLs elicit different antigen-specific CTL responses from patient to patient, and that the CTL populations generated by APL stimulation are capable of effectively killing tumors. Thus these agonist APLs might be considered “conditional” superagonist ligands. Using unique tumor epitope-specific CTL clones in the initial screen that other potential superagonist peptides can be identified. Panels of potential tumor-associated superagonist peptides may be assembled, to ensure that one or more APLs are effective at generating potent anti-tumor CTL responses from a given patient.

Efficacy

To determine how well the identified agonist APLs could prospectively generate tumor epitope-specific CTL populations from peripheral blood mononuclear cell (PBMC) of tumor patients, the APLs were used to stimulate different patient PBMC samples under standard in vitro conditions. Preferentially, cultures of PBMC are treated with the mutagenized epitope and incubated for at least one week. CTLs can readily be measured using ordinary methods. For example, cells can be stained with FITC-labeled anti-CD8 antibodies and APC-labeled HLA-matched complexes and analyzed by flow cytometry.

The ability of an APL to generate CD4 T cells from PBMC of tumor patients is also a measure of the efficacy of the mutagenized epitope.

It may be necessary to probe a panel of APLs since the ability of a single APL to stimulate cells of every patient having the specific tumor cannot be assumed at the outset of measurements.

One aspect of the utility of the APLs lies in their ability to stimulate T cells of a cancer patient ex vivo or in vivo. The stimulated T cells are effector and regulator CD4⁺ cells, including Th1, Th2, Th9 and/or Th17 cells. The stimulation can involve use of the APLs as purified peptides, or as intracellular products of APL minigenes. APL minigenes may also be expressed as a string of beads, i.e., multiple CTL genes within the same expression vector, or as part of a T helper protein as described in Fomsgaard et al., 1999 Vaccine 18:681-91; Ann et al., 1997 J Virol 1192-302; Toes et al., 1997 Proc Natl Acad Sci 94:14660-65; Gao et al., 2006 Vaccine 24:5491-97, hereby incorporated by reference in their entirety.

The potential use for these novel antigenic peptides includes their use in anti-tumor vaccine studies; use in adoptive immunotherapy to generate a wider array of anti-tumor CD4⁺ T cell clonotypes; the ability to alter the phenotype of T regulatory cells in order to more effectively activate anti-tumor CD4⁺ T cells.

EXAMPLES Example 1

Oligonucleotides were designed to have a complimentary 5′ KpnI site and a complimentary 3′ PstI site. The sequences of the saturation mutagenesis sense strands of the MART-1₂₆₋₃₅ positional oligonucleotides are shown in Table 5 (each sense strand has a corresponding mutant antisense strand):

TABLE 5 MART- SEQ 1₂₆₋₃₅ ID Sense strand of MART-1₂₆₋₃₅ positional Library NO: Saturation Mutagenesis Oligonucleotides P1 352 CATCGAGGGAAGGNNNCTCGCCGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA P2 353 CATCGAGGGAAGGGAGNNNGCCGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA P3 354 CATCGAGGGAAGGCAGCTCNNNGGAATCGGCATTCTGACCGTTTAATGAATTCTGCA P4 355 CATCGAGGGAAGGCAGCTCGCCNNNATCGGCATTCTGACCGTTTAATGAATTCTGCA P5 356 CATCGAGGGAAGGCAGCTCGCCGGANNNGGCATTCTGACCGTTTAATGAATTCTGCA P6 357 CATCGAGGGAAGGCAGCTCGCCGGAATCNNNATTCTGACCGTTTAATGAATTCTGCA P7 358 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCNNNCTGACCGTTTAATGAATTCTGCA P8 359 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCATTNNNACCGTTTAATGAATTCTGCA P9 360 CATCGAGGGAAGGCAGCTCGCCGGAATCGGCATTCTGNNNGTTTAATGAATTCTGCA NNN represents totally randomized codons, any one of sixty-four codons. In a given positional library consisting of 100 mutant oligonucleotide pairings, each codon has high likelihood of being represented.

Variant polypeptide sequences are listed in Table 6.

TABLE 6 Designation Sequence SEQ ID NO Mart-1 EAAGIGILTV 361 A27L ELAGIGILTV 362 E26G GLAGIGILTV 363 E26S SLAGIGILTV 364 L33M ELAGIGIMTV 365

Similarly, nucleotides encoding variant sequences of NY-ESO₁₅₇₋₁₆₅ were synthesized that encoded the following sequences (Table 7).

TABLE 7 Designation Sequence SEQ ID NO: NY-ESO-1₁₅₇₋₁₆₅ WT SLLMWITQVC 366 NY-ESO-1₁₅₇₋₁₇₀ WT SLLMWIITQCFLPVF 367 W161I (NYII-51) SLLMFITQCFLPVF 368 W161F SLLMFITQCFLPVF 369 I162R SLLMWRTQCFLPVF 370 I162M SLLMWMTQCFLPVF 371 I162Q (NYII-6Q) SLLMWQTQCFLPVF 372 1162V SLLMWVTQCFLPVF 373 Q164S (NYII-8S) SLLMWITSCFLPVF 374 F170W (NYII-14W) SLLMWITQCFLPVW 375 NY-ESO-1 C165V SLLMWITQVF 376 Synthetic polypeptides having these sequences were suspended in DMSO.

Example 2

The saturation mutagenesis oligonucleotides were cloned into the expression vector pQE40 (Qiagen). The plasmids were transformed into E. coli (M15 pREP). Mini-gene products were expressed as fusion proteins containing 6×-histidine tags. Following recombinant protein induction, bacteria were lysed with 8M Urea, pH 8.0. Lysate was harvested and applied to Mg²⁺ coated paramagnetic beads (Talon beads, Dynal), which bind specifically to 6×-histidine.

For saturation mutagenesis libraries, bacterial clones were cultured individually in wells of 96-well plates.

Melanoma cell lines A375 and MeI 526, CTL clones and the TAP-deficient cell line T2 were maintained in RPMI 1640, containing 25 mM HEPES, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin and 10% human serum from normal donors. Dendritic cells were prepared from adherent monocytes, isolated from the PBMC of HLA-A2⁺ healthy donors. IL-4 (500 U/mL; R&D Systems, Minneapolis, Minn.) and GM-CSF (800 U/mL; Amgen, Thousand Oaks, Calif.) were added to the monocytes to promote their differentiation into dendritic cells. MART-1₂₆₋₃₅₋specific CTL clones were generated as described by Li et al., 2005. J Immunol 175:2261-69, hereby incorporated by reference in its entirety. PBMC used in this study were obtained from HLA-A2⁺ melanoma patients.

Example 3 Saturation Mutagenesis APL Screen

Following the isolation of the recombinant mini-gene APL products on Talon beads, the bead-bound products were “fed” to 100,000 immature dendritic cells. Following a 4-hour incubation at 37° C., 100,000 MART-1₂₆₋₃₅-specific CTL clones were added to DC/bead preparations. Following a 12-hour incubation at 37° C., the supernatant was harvested and assayed for the concentration of IFN-γ induced by the APL clones. Anti-IFN-γ antibodies (Endogen) used in the sandwich ELISA were used at 1 μg/ml in PBS/0.1% BSA.

Variant MART-126-35 agonist peptides identified using mutagenesis APL screening and their corresponding DNA sequences are shown in Table 8.

TABLE 8 Amino Acid SEQ ID Designation Sequence SEQ ID NO DNA Sequence NO MART-1₂₆₋₃₅ EAAGIGILTV 395 NA A27L ELAGIGILTV 396 NA E26G GLAGIGILTV 397 ggactcgccggaatcggcattctgacc 377 E265 SLAGIGILTV 398 tcactcgccggaatcggcattctgacc 378 E26S SLAGIGILTV 399 tcgctcgccggaatcggcattctgacc 379 E265 SLAGIGILTV 400 agtctcgccggaatcggcattctgacc 380 L33M ELAGIGIMTV 401 gagctcgccggaatcggcatgctgacc 381

Example 4 In Vitro PBMC Stimulations with Analog Peptides and Tetramer Staining

On day 0, monocyte-derived dendritic cells were pulsed with 1 μM of each MART-1₂₆₋₃₅ analog peptide for 2 hours at 37° C. The DCs were washed and added to 500,000 HLA-A2⁺ PBMC from melanoma patients at a 1:20 ratio in 24-well plates. On day 2, 12.5 U/ml of IL-2, 5 ng/ml IL-7, 1 ng/ml IL-15, and 10 ng/ml of IL-21 were added to each culture. Cytokines were replenished every 2-3 days for 1-week. Following the 1-week primary stimulation, cultures were re-stimulated with 1×10⁶ irradiated monocytes pulsed with 10 μM of the peptide used in the primary stimulation. IL-2, IL-7 and IL-15 were added to secondary stimulations on day 2. Cytokines were replenished every 2-3 days. 500,000 cells from each culture were stained with APC-labeled anti-CD8 antibody (Caltag Lab, Burlingame, Calif.) and PE-labeled MART-1₇₆₋₃₅ HLA-A2.1 tetramers. Stained cells were analyzed using FACScalibur flow cytometer and CellQuest (BD PharMingen) and analyzed using FlowJo software v8.5 (Tree Star, San Carlos, Calif.). Cells were stained with tetramers in 25 μl of 2% FCS/BSA for 1 hour at room temperature, followed by anti-CD8 antibody for 15 minutes at 4° C.

Example 5 Generation of MART-1₂₆₋₃₅ Polyclonal Cell Lines

Following in vitro peptide stimulation of HLA-A2⁺ PBMC from melanoma patients MelPt-B, MelPt-C, MelPt-D, MelPt-F and a healthy donor (Healthy-1) MART-1₂₆₋₃₅ tetramer and CD8 positive cells were sorted and isolated on BD FACSaria. Isolated cells were replicated using 30 ng/ml anti-CD3 antibody (OKT3) and IL-2 at 50 U/ml in the presence of irradiated feeder PBMC and LCL for 2 weeks. IL-2 was replenished every 2-3 days. Following the stimulation, cultures were stained for the generation of MART-1₂₆₋₃₅ tetramer and CD8 positive cell populations. The polyclonal cell lines were tested for lytic activity and TCR Vβ usage (MelPt-C only), as described in Example 6.

Example 6 In Vitro Cytotoxicity Assay

Target cells were labeled with 100 μCi of ⁵¹Cr and co-cultured with effector cells for 4 hours at 37° C. plus 5% CO₂. Targets were melanoma cell lines A375 (HLA-A2⁺/NY-ESO-1⁺) and MeI 526 (HLA-A2⁺/MART-1⁺), and T2 cells pulsed with 1 μM of MART-1₂₆₋₃₅ (positive control) or NY-ESO-1₁₅₇₋₁₆₅ (negative control). Effector cells were MART-1₂₆₋₃₅-tetramer positive polyclonal cell lines generated with either A27L, E26S, or L33M peptides. Assays were performed in triplicate at a 50:1, 25:1 or 12.5:1 effector to target ratio. Released ⁵¹Cr was measured with a gamma scintillation counter and percent specific lysis was determined by using the formula: percent specific release=(experimental release-spontaneous release)/(maximum release-spontaneous release).

Example 7 TCR Spectratype Analysis

TCR Vβ spectratype analysis was carried out by the Immune Monitoring Laboratory at Fred Hutchinson Cancer Research Center. Briefly, cDNA was generated from 1×10⁶ MART-1₂₆₋₃₅ tetramer staining polyclonal cell lines. Multiplex Vβ PCR primers were then used to amplify the variable regions of the complementarity-determining region 3 (CDR3) of the TCR β chain. Sequence analysis to determine the Vβ usage of the TCRs was conducted with GenScan.

Example 8 Mart-1₂₆₋₃₅ Specific CTL Clones can Detect Enhanced CTL Epitopes as Reflected by IFN-γExpression

To identify superagonist APLs, a novel genetic system was discovered and developed. This system employs saturation mutagenesis of agonist peptide-encoding oligonucleotides, which when expressed in E. coli will contain position specific single amino acid substitutions. The positional libraries are designed such that the codon of interest is totally randomized (NNN), resulting in a pool of oligonucleotides which contains every given codon sequence. This mutagenesis approach might be likened to a slot machine which contains three positions (a codon) and each position has the same 4 possibilities (A, C, G or T). When pulled, there is a 1 in 64 chance of getting any combination of 3. If pulled 100 times there is a high probability that every sequence will be represented (80% certainty, according to a Poisson distribution). Here, the 100 pulls represent 100 bacterial colonies, each containing a different mutant agonist peptide-encoding oligonucleotide. When cloned and expressed, each amino acid should be represented in a library of 100, with 80% certainty, according to a Poisson distribution. A positional library can be generated for each position (amino acid) of the target peptide. The APL min-gene constructs are fused to a 6×-histidine tag, and can easily be separated from bacterial proteins on Co²⁺-coated paramagnetic beads. APLs are screened for the ability to activate epitope-specific CTL clones following cross-presentation of the bead-bound ligand on class I MHC molecules by immature dendritic cells (DC).

To validate this system and to verify that it was sensitive enough to detect our model tumor-associated HLA-A2 restricted antigenic peptide, MART-1₂₆₋₃₅, as well as an APL superagonist epitope of MART-1₂₆₋₃₅, called MART-1₂₆₋₃₅A27L (henceforward referred to as A27L), oligonucleotides encoding the appropriate peptide sequences were cloned, expressed and assayed for the ability to activate antigen specific CTL clones as described in materials and methods. The CTL clone used in this assay, called M26-H1, is specific for MART-1₂₆₋₃₅, and expresses TN-γ in response to HLA-A2/MART-1₂₆₋₃₅ complexes. Here, the TN-γ response elicited by the recombinant unmodified MART-1₂₆₋₃₅ cross-presented construct is significantly higher than that elicited by the HLA-A2 restricted negative control, NY-ESO-1₁₅₇₋₁₆₅ (FIG. 1). Further, the IFN-γ response elicited by the recombinant superagonist APL, A27L, was more than 2-fold higher than that elicited by the recombinant wild type construct. Yet, the activation of M26-H1 by the unmodified MART-1₂₆₋₃₅ construct was clearly distinguishable from that elicited by the HLA-A2 restricted negative control construct, NY-ESO1₁₅₇₋₁₆₅. These results suggest that the HLA-A2 cross-presented recombinant ligands are sufficient to elicit detectable antigen-specific responses from CTL clones, and also that superagonist APLs can be distinguished based on an increase in TN-γ expression, relative to the wild type CTL ligand.

Example 9 Saturation Mutagenesis can Effectively Generate Random Amino Acids in the Parental Antigenic Peptide from which Enhanced Agonist APLS can be Identified

The saturation mutagenesis APL library screen depends on 200 μl bacterial expression cultures in 96-well plates. FIG. 1 shows that cross-presented recombinant ligands can be detected by antigen-specific CTL. However, in that experiment recombinant proteins were produced at high concentrations in 5 ml cultures. To determine whether the recombinant protein produced in these significantly smaller cultures would be sufficient to reflect detectable and varying degrees of activation, a position 2 (P2) library of MART-1₂₆₋₃₅ (EXAGIGILTV) was constructed. By screening this library, in addition to determining if 200 μl cultures produce sufficient concentrations of recombinant protein previously identified superagonist APLs, including A27L could be identified from among 88 unique mutant APL clones. The P2 library screen (FIG. 2), using the CTL clone M26-H1, clearly shows that the wild type recombinant ligand MART-1₂₆₋₃₅ elicits significantly more IFN-γ than the negative control. Furthermore, the APL clones from the library that contained leucine residues at P2 (A27L), elicited significantly more IFN-γ expression in comparison to the wild type ligand. Amino acid content was determined from replicated glycerol stock of the P2 bacterial library. Interestingly, APL clones containing methionine residues at P2 also elicited greater IFN-γ expression than wild type MART-1₂₆₋₃₅, although not as great as that elicited by the leucine containing APLs, A27L. Like A27L, A27M is a superagonist APL of MART-1₂₆₋₃₅. Thus, 200 μl bacterial cultures produce sufficient concentrations of the recombinant ligands to be detected in this screen. Also, superagonist APLs can be identified in a library of at least 88 unique APL clones.

Example 10 Putative Enhanced CTL Epitopes of Mart-1₂₆₋₃₅A27L are Identified in APL Library Screens

On the basis of previous results demonstrating that superagonist APLs can be identified and defined using the saturation mutagenesis screen, remaining positional libraries of MART-1₂₆₋₃₅, were screened using similar methods. Because a potent superagonist APL of MART-1₂₆₋₃₅ has already been identified in A27L, A27L was used as the basis for a mutational strategy. That is, leucine in position 2 was constant, while other positions were mutated independently. This would allow superagonist APLs to be identified that are more effective than A27L.

The APL libraries were screened with two different high avidity MART-1₂₆₋₃₅-specific CTL clones. A high avidity TCR is defined as having the ability to recognize tumor cells that express both MART-1 and HLA-A2 class I molecules. The vast majority of the MART-1₂₆₋₃₅ derivative mutant peptide clones screened from each of the positional libraries were not as effective as A27L at activating the MART-1₂₆₋₃₅₋specific CTL clone (FIG. 3). However, several clones from the P1, P3 and P8 libraries appeared to work similarly as well as the A27L recombinant construct. The initial screen was conducted by screening two unique APL library clones simultaneously in a single well. While this approach allows twice as many APL clones to be screened, the potency of any agonist APL in the pool is potentially underestimated in the initial screen.

Agonist candidates were selected and re-screened based on their ability to elicit more or comparable levels of IFN-γ from M26-H1 in the initial screen (FIG. 3B). When tested independently, both of the clones from the P3 libraries elicited less IFN-γ expression from the MART-1₂₆₋₃₅-specific CTL clone, relative to A27L. When re-screened independently, it was apparent that only one of the two mutant peptide clones from the P1 and P8 wells was responsible for the increased IFN-γ expression. The DNA encoding these putative MART-1₂₆₋₃₅ agonist peptides was prepared from the duplicated bacterial glycerol stocks. The enhancing mutations for the P1 putative agonists contained either glycine (E26G) or serine (E26S) residues at P1 instead of the naturally occurring glutamate residue. The P8 putative agonist contained a methionine residue (L33M) at position 8 rather than the naturally occurring leucine residue. No additional putative agonists were identified from the library screens using the second CTL clone, M26-H2.

Example 11 MART-1₂₆₋₃₅ Agonist Peptides Display a Differential Capacity to Activate Different MART-1₂₆₋₃₅₋specific CTL Clones

To analyze the putative superagonist APLs on a molar basis, individual peptides were synthesized at greater than 90% purity. To determine whether these APLs would be similarly recognized by unique MART-1₂₆₋₃₅-specific CTL clones, the APLs were tested against four clones bearing unique T cell receptors (TCR). These included two high avidity CTL clones (M26-H1 and M26-H2) and two low avidity CTL clones (M26-L1 and M26-L2) (FIG. 4). Low-avidity TCR is here defined as having the ability to respond HLA-A2 positive peptide-pulsed target cells but not to cells displaying naturally processed and presented determinants from HLA-A2/MART-1 positive tumors. Low-avidity T cells have the potential to mediate antigen-specific cell and tissue destruction.

FIG. 4 panel A shows that each of the newly identified agonist peptides is similarly effective in activating M26-H1—the high-avidity CTL clone used in the initial screen (as described in FIG. 3) as compared to MART-1₂₆₋₃₅ superagonist peptide, A27L. A similar pattern of activation was found when the identified agonist peptides are used to stimulate the CTL clone M26-H2. In contrast to the above results, the low-avidity MART-1₂₆₋₃₅-specific CTL clones yielded widely divergent results in response to different agonist peptides. For example, while the CTL clone M26-L1 recognizes the peptide E26S more than 100-fold better than A27L (based on half-maximal activation), the CTL clone M26-L2 recognizes A27L better than it does E26S. Similarly, while L33M is scarcely recognized by the CTL clone M26-L1, it is the most effective agonist for activating M26-L2. Thus, these analogs might be considered “conditional” agonists, as they do not elicit generalized patterns of activation among unique antigen-specific clonotypes.

Example 12 MART-1₂₆₋₃₅ APLs Demonstrate Patient-Specific Enhanced Generation of MART-1₂₆₋₃₅ CTL Populations from the PBMC of Melanoma Patient Donors

To determine how well the identified agonist APLs could prospectively generate MART-1₂₆₋₃₅-specific CTL populations from melanoma patient peripheral blood mononuclear cell (PBMC) preparations, the APLs were used to stimulate eight different patient PBMC samples under standard in vitro conditions (Table 9).

TABLE 9 Patient A27L E26G E26S L33M MelPt-A 3.14 (1) 1.68 (0.53) 3.36 (1.07) 0.98 (0.31) MelPt-B 2.97 (1) 1.31 (0.44)  4.3 (1.45)  7.7 (2.6) MelPt-C 40.6 (1) 45.6 (1.12) 15.6 (0.38) 41.1 (1.02) MelPt-D 0.65 (1) 1.73 (2.66) 3.43 (5.27) 2.07 (3.1) MelPt-E 1.77 (1) 8.42 (4.75) 6.88 (3.88) 24.2 (13.67) MelPt-F 5.45 (1) 3.35 (0.61) 3.72 (0.68) 3.07 (0.56) MelPt-G 33.4 (1) 1.89 (.06) 1.75 (.05) 2.37 (.07) MelPt-H 1.24 (1) 2.03 (1.63) 1.31 (1.06) 2.77 (2.2)

These results show that MART-1₂₆₋₃₅ APLs exhibit differential capacities to generate MART-1₂₆₋₃₅-specific CTL populations from the PBMC of different melanoma patient donors. APLs were used to stimulate PBMC cultures in vitro. Following a one-week secondary stimulation cells were stained with FITC-labeled anti-CD8 antibodies and APC-labeled HLA-A2/MART-1₂₆₋₃₅ tetramers and analyzed by flow cytometry. Values are given as percent tetramer positive relative to a negative control. The fold difference relative to A27L is indicated in parentheses. Differences of more than two-fold are indicated in bold.

One week following the second in vitro stimulation, cultures were stained with the wild-type MART-1₂₆₋₃₅/HLA-A2 tetramer. Similar to the observations made using different MART-1₂₆₋₃₅-specific CTL clones, none of the peptide ligands were universally effective in generating MART-1₂₆₋₃₅-specific CTL populations from all patient PBMC samples (FIG. 5). Any given APL was more or less effective in generating antigen-specific CTL from any given patient PBMC sample. For example, while the agonist peptide E265 is the least effective at generating MART-1₂₆₋₃₅-specific CD8 positive populations from the PBMC of MelPt-C (3-fold<A27L), it is the most effective APL for generating such T cell populations from MelPt-D (5-fold>A27L) Similarly, whereas the agonist peptide L33M is 14-fold more effective than A27L in generating of MART-1₂₆₋₃₅-specific CD8 positive populations from the PBMC of MelPt-E, it is 14-fold less effective than A27L in generating MART-1₂₆₋₃₅-specific CD8 populations from the PBMC of MelPt-G. These findings demonstrate that any one CTL ligand may not be effective at generating antigen-specific CTL populations from the PBMC of any given patient; and show the importance of establishing a panel of potential superagonist APLs.

Example 13 CD8 Positive MART-1₂₆₋₃₅-Specific Polyclonal Cell Lines Generated with the Identified MART-1₂₆₋₃₅ Agonist APLS can Kill HLA-A2⁺ Tumors Expressing Endogenous MART-1

The use of altered peptide ligands poses the risk of generating antigen-specific T cells which display relatively low anti-tumor functional avidity. To determine whether the MART-1₂₆₋₃₅-specific CTL that were generated with these novel MART-1₂₆₋₃₅ agonist peptides were of sufficient functional avidity to kill HLA-A2/MART-1 positive tumor targets, polyclonal lines of CD8 positive MART-1₂₆₋₃₅ tetramer-staining cells were established from the PBMC of MelPt-B, MelPt-C, MelPt-D, MelPt-F or a healthy donor (Healthyl), stimulated with either A27L, E265 or L33M agonist peptides. These cell lines were screened for reactivity to unmodified MART-1₂₆₋₃₅ peptide pulsed HLA-A2 positive targets and to HLA-A2/MART-1 positive tumor targets at varying effector to target ratios in a standard chromium release assay (Table 10).

TABLE 10 Tumor specific lysis by CTL generated with MART₁₂₆₋₃₅ peptide analogs Percentage Specific Lysis from polyclonal CTL lines generated with the indicated peptide MART-1₂₆₋₃₅A27L MART-1₂₆₋₃₅E26S MART-1₂₆₋₃₅L33M T2 + T2 + T2 + Patient E/T T2 M26 A375 Mel526 T2 M26 A375 Mel526 T2 M26 A375 Mel526 MelPt-B 50 ND ND ND ND 0^(c) 40 6 58 2 63 13 80 25 ND ND ND ND 0 31 4 45 2 52 12 70 12.5 ND ND ND ND 2 21 8 30 4 41 9 58 MelPt-C 50 9 73 3 35 2 87 8 41 9 91 0 58 25 10 52 5 30 7 66 5 32 12 84 2 52 12.5 9 42 2 25 1 54 2 25 11 76 5 45 MelPt-D 50 0 50 10 32 3 87 8 41 14 90 2 57 25 2 42 10 30 8 65 7 32 11 84 4 54 12.5 5 35 9 25 1 54 6 25 8 75 6 45 MelPt-F 50 6 92 5 65 ND ND ND ND 23 70 2 44 25 3 81 5 50 ND ND ND ND 21 72 3 42 12.5 2 73 5 43 ND ND ND ND 22 65 5 38 Healthy1 50 2 52 3 58 0 34 9 28 2 57 13 65 25 4 42 7 42 0 26 5 15 6 49 13 57 12.5 1 31 6 35 0 18 8 10 5 38 10 44 “ND” is not done. T2 is a TAP-deficient cell line that expresses peptide-unbound HLA-A2 molecules unless pulsed extracellularly. Here, T2 was pulsed with NY-ESO-1₁₅₇₋₁₆₅ unless indicated otherwise. M26 is an abbreviation for the unmodified MART-1₂₆₋₃₅ peptide. Numbers represent the percentage specific lysis obtained from each target. T375 is a HLA-A2 positive/MART-1 negative cell line.

The results illustrate that the CTL populations that were generated from each PBMC source with either of the altered peptide ligands can kill targets that display wild-type MART-1₂₅₋₃₅ in the context of HLA-A2, and recognize the epitope with sufficient affinity to kill tumors expressing MART-1.

To determine whether unique or shared MART-1₂₅₋₃₅₋specific CTL clonotypes were generated with each of the peptide ligands (A27L, E26S and L33M), spectratype analysis was performed on CTL lines derived from MelPt-C PBMC to determine their Vβ TCR usage. Results showed that the agonist peptides A27L, E26S and L33M generated CTL populations that primarily (>90%) utilized TCR Vβ24, Vβ8 and Vβ3, respectively. This suggests that the different analog peptides preferentially generate specific TCR utilizing CTL subsets. Taken together, these results demonstrate the ability of the identified APLs to elicit MART-1₂₆₋₃₅-specific CTL responses that are capable of directly killing MART-1 expressing tumors, and suggest that unique MART-1₂₅₋₃₅-specific TCR subpopulations are being preferentially generated by the different MART-1₂₆₋₃₅ analog peptides.

Example 14 NY-ESO APLs

Using the methods of Examples 2-8 were used to generate enhanced agonist APLs. Results of a library screen are shown in FIG. 6. Clones showing activity were sequenced. Variant sequences with the most activity correspond to amino acid sequences of SEQ ID NOS:368-375.

Using the methods of Example 11 to analyze the putative superagonist APLs on a molar basis, individual peptides were synthesized at greater than 90% purity. To determine whether these APLs would be similarly recognized by unique NY-ESO-1-specific CTL clones, the APLs were tested against ten clones bearing unique TCR. Each of the newly identified agonist peptides is similarly effective in activating CTL clones used in the initial screen in comparison to wild-type NY-ESO-1₁₅₇₋₁₇₀ superagonist peptide. Different patterns of stimulation are obtained with different CTL clones. Specific CTL clones yielded widely divergent results in response to different agonist peptides. Similar to results obtained with MART superagonist peptides, these NY-ESO-1 analogs might be considered “conditional” agonists, as they do not elicit generalized patterns of activation among unique antigen-specific clonotypes.

The NY-ESO-1 C165V APL (SEQ ID NO:376) was compared to wild-type NY-ESO-1₁₅₇₋₁₆₅ SEQ ID NO:377 in effectively producing CTL from PBL. FIG. 7 shows that the variant peptide had a higher avidity than the wild type sequence to a CD-8⁺ population.

FIG. 8 show the ability of several NY-ESA-II APL to stimulate CD-4⁺ fractions (FIG. 8A) and PBMC (FIG. 8B) and. Results showed that NY-ESO-1 APLs 1162Q, Q164S, and F170W were the most effective in stimulating CD-4⁺ cells.

NY-ESO-1₁₅₇₋₁₇₀ agonist peptides identified using mutagenesis APL screen and their corresponding DNA sequences are shown in the following Table 11.

TABLE 11 SEQ SEQ Amino Acid ID ID Designation Sequence NO DNA Sequence NO NY-ESO-1₁₅₇₋₁₇₀ SLLMWITQCFLPVF 402 NA W161I SLLMIITQCFLPVF 403 agcctgctgatgatcattacccagtgctttctgccggtgttttaa 382 W161I SLLMIITQCFLPVF 404 agcctgctgatgattattacccagtgctttctgccggtgttttaa 383 Q164S SLLMWITSCFLPVF 405 agcctgctgatgtggattacctcatgctttctgccggtgttttaa 384 F170W SLLMWITQCFLPVW 406 agcctgctgatgtggattacccagtgctttctgccggtgttttgg 385 W161F SLLMFITQCFLPVF 407 agcctgctgatgtttattacccagtgctttctgccggtgttttaa 386 I162R SLLMWRTQCFLPVF 408 agcctgctgatgtggaggacccagtgctttctgccggtgttttaa 387 I162M SLLMWMTQCFLPVF 409 agcctgctgatgtggatgacccagtgctttctgccggtgttttaa 388 I162Q SLLMWQTQCFLPVF 410 agcctgctgatgtggcaaacccagtgctttctgccggtgttttaa 389 I162V SLLMWVTQCFLPVF 411 agcctgctgatgtgggtgacccagtgctttctgccggtgttttaa 390 Q164S SLLMWITSCFLPVF 412 agcctgctgatgtggattacctcttgctttctgccggtgttttaa 391 

1. Use of a compound for the treatment of a patient with a tumor, wherein the compound comprises a peptide comprising a tumor epitope selected from the group consisting of SEQ ID NOS:361-376, and 392-401.
 2. The use according to claim 1, wherein the tumor epitope is selected from the group consisting of SEQ ID NOS:361-376.
 3. The use according to claim 1, wherein the tumor epitope is NYESO-I₁₅₇₋₁₆₅ (SEQ ID NO:366), NYESO-II₁₅₇₋₁₇₀ (SEQ ID NO:367) or MART-1₂₆₋₃₅ (SEQ ID NO:361).
 4. The use according to claim 1, wherein the tumor epitope is NYESO-I₁₅₇₋₁₆₅ (SEQ ID NO:366).
 5. The use according to claim 1, wherein the tumor epitope is NYESO-II₁₅₇₋₁₇₀ (SEQ ID NO:367).
 6. The use according to claim 1, wherein the tumor epitope is MART-1₂₆₋₃₅ (SEQ ID NO:361).
 7. The use according to claim 1, wherein the compound comprises a multiplicity of said peptides having at least two sequences selected from the group consisting of SEQ NOS:361-376, and 392-401.
 8. The use according to claim 1, wherein the compound comprises a multiplicity of said peptides having at least two sequences selected from the group consisting of SEQ ID NOS:362-365 and 368-376.
 9. The use according to claim 1, wherein T cells of said patient react with said compound.
 10. The use according to claim 1, wherein the compound is an antitumor vaccine.
 11. The use according to claim 1, wherein the compound is produced by expressing a gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:382-391 and 377-381.
 12. The use according to claim 11, where gene is expressed in human cells.
 13. The use according to claim 12, wherein the gene is expressed in cells of a melanoma patient.
 14. The use according claim 1, wherein the patient has a melanoma. 15.-24. (canceled)
 25. A method of treating a patient with a tumor, comprising administering a pharmaceutical composition to said patient, wherein said pharmaceutical composition comprising a peptide comprising a sequence selected from the group consisting of SEQ ID NOS:361-376
 26. The method according to claim 25, consisting of combining the APL with cells of a patient.
 27. The method according to claim 25, wherein the tumor epitope comprises a sequence selected from the group consisting of SEQ ID NOS:361-376.
 28. The method according to claim 27, wherein the tumor epitope comprises a sequence of NYESO-I₁₅₂₋₁₆₅ (SEQ ID NO:366), NYESO-II₁₅₂₋₁₇₀ (SEQ ID NO:367) or MART-1₂₆₋₃₅ (SEQ ID NO:361).
 29. The method according to claim 25, wherein the APL is used in adoptive immunotherapy to generate activated T cells.
 30. The method of claim 29, wherein the superagonist APL is used to activate anti-tumor T cells.
 31. The method of claim 30, wherein the cells of the patient are ex vivo. 